The present invention is related to new metal chelator resins and their manufacture process.
Metal Chelate Affinity Chromatography (MCAC) (also denoted Immobilised Metal ion Affinity Chromatography (IMAC)) using affinity immobilised metal resins introduced by Porath et al. (Nature, 258, 589 (1975) and used for the purification of proteins which contain neighbouring histidine residues, has now become a powerful and versatile tool for the purification of natural and recombinant 6xc3x97 His-tagged (or not) proteins and peptides.
The ligand used by these authors was the iminodiacetic acid (IDA). Electron paramagnetic resonance and absorption spectra studies have demonstrated that IDA is a tridentate ligand and the configuration of the complex IDA-M2+ (1:1) with M2+=bivalent metal ions, is a square or tetrahedral one (R. Dallocchio et al.; J. Coord. Chem., 25, 265 (1992). This explains why immobilised IDA can form a stable complex with the ion Cu2+ and Zn2+, but not with other heavy metal ions which need the octahedral configuration for a stable form.
It is also known that histidine is the only xcex1-aminoacid capable of forming octahedral complexes with different polyvalent metal ions as follows: Hisxe2x80x94M2+xe2x80x94His (B. Rao et al.; J. Inorg. Nucl. Chem.; 33, 809 (1971); M. M. Harding et al.; Acta Cryst., 16, 643 (1963)):
Each histidine gives 3 coordination bonds to the M2+ i.e. the 3-N group of the imidazole ring and the NH2 and COOH groups of the aminoacid; the 1-NH group of the imidazole ring does not participate in the formation of the complexes (C. C. Mc Donald et al.; JACS, 85, 3736 (1963)).
The complex formation is stereoselective (J. H. Rituma et al.; Recueil, 88, 411 (1969).
The complex chemistry of histamine and imidazole has been described (W. R. Walker et al.; J. Coord. Chem., 3, 77 (1973); Aust. J. Chem., 23, 1973 (1970)).
Furthermore, Single-crystal X-ray analysis (Simon H. Whitlow, Inorg. Chem., 12, 2286 (1973)) and Infrared Spectra studies (Y. Tomita et al.; JACS, 36, 1069 (1963) and J. Phys. Chem., 69, 404 (1965)) have demonstrated that Trisodium Nitrilotriacetate (Na3NTA) is a tetradentate ligand for different polyvalent metal ions M2+ and the corresponding complexes NTA-M2+ have an octahedral configuration:
At pH 5.5-10.0, NTA may be a mixture of HN+(CH2xe2x80x94COOxe2x80x94)3 and N(CH2xe2x80x94COOxe2x80x94)3.
Only the carboxylate and uncharged N groups participate in the coordination bonding. The carboxylic and charged N groups do not participate in such linkages.
The NTA derivatives immobilised on Agarose introduced by E. Hochuli et al. (J. Chromatogr., 411, 177 (1987)) and U.S. Pat. No. 4,877,830 (1989) can be therefore, an interesting method for the purification of histidine containing proteins. Their ligands are H2Nxe2x80x94(CH2)nxe2x80x94CH(COOH)xe2x80x94N (CH2xe2x80x94COOH)2 (n=2,4) and the resulting resins are: Resin-NHxe2x80x94(CH2)nxe2x80x94CH(COOH)xe2x80x94N (CH2xe2x80x94COOH)2 (n=2,4).
The present invention aims to provide new chelator resins having improved characteristics over the compounds of the state of the art and being suitable for metal chelate affinity chromatography.
The present invention is also related to the preparation process of such resins.
The present invention is related to an easy, rapid and inexpensive manufacture method of novel resins for IMAC and to said resins being hereafter called Pentadentate chelator (PDC) resins, which advantageously afford 5 coordination bonds to the M2+ ions. Said coordination bonds may result in an improved stability of the obtained octahedral complexes and one coordination site is free for interaction and selective binding to accessible cysteine/histidine residues and chiefly histidine containing biomolecules that are preferably selected from the group consisting of proteins or peptides.
Furthermore, said PDC resins are able to chelate with different polyvalent metal ions including Cu2+, Ni2+, Zn2+ and Co2+ to give the corresponding metal chelate resins hereafter called Cuxe2x80x94PDC, Nixe2x80x94PDC, Znxe2x80x94PDC and Coxe2x80x94PDC respectively. These four resins will be thereafter used for the purification of histidine containing natural and recombinant proteins or peptides.
The present invention is also related to said PDC resins, for which the proteins cannot enter into the pores of the resin (molecular weights of proteins are greater than 5000 Daltons, by definition). Preferably, said resin is PDC-Sephadex(copyright) G-25 (obtained from Sephadex(copyright) G-25, Pharmacia, Uppsala, Sweden).
In addition, the binding of histidine containing proteins to the chelated metals depends on the complex Metal-PDC resins and the accessibility of histidine residues which in turn, depends on the configuration of the proteins of interest. Therefore, there is no universal rule that will predict the order of magnitude of binding of histidine containing biomolecules to Cu2+, Ni2+, Zn2+ and Co2+.
The present invention is also related to a PDC KIT consisting of four separate columns Cuxe2x80x94PDC, Nixe2x80x94PDC, Znxe2x80x94PDC and Coxe2x80x94PDC, that determine the most appropriate metal chelate resin suitable for the purification of natural and recombinant biomolecules, preferably selected from the group consisting of proteins or peptides.
The Cu-PDC resins according to the invention are used as universal supports for immobilising covalently proteins using a water-soluble carbodiimide and also as concentration resins to reduce the volume of a protein solution.
A last aspect of the present invention concerns the use of the pentadentate chelator (PDC) resins, and especially the PDC-Sephadex(copyright) G-25, according to the invention to obtain water and buffers free of polyvalent metal ions. In particular, the PDC-Sephadex(copyright) G-25 according to the invention is useful for preparing xe2x80x9cmetallo-proteinsxe2x80x9d free of heavy metal ions or proteins free of heavy metal ions after the Immobilised Metal ion Affinity Chromatography steps.